To investigate the ability of IGF-1 and insulin to suppress the programmed cell-death, apoptosis was induced in rabbit lens epithelial cells by treatment with staurosporine (10 ng/mL), a common inducer of cell apoptosis. Incubation of rabbit lens epithelial cells with staurosporine for 2–3 hours produced significant apoptosis within 8–10 hours. Apoptosis was assessed by several criteria: the characteristic ladder-like DNA fragmentation, nuclear staining with Hoechst fluorescent reagent, and cell morphology. As shown in
Figure 1A , staurosporine induced significant breakdown in cellular DNA. The presence of various concentrations of IGF-1 (5–50 nM) suppressed the staurosporine-induced apoptosis in a dose-dependent manner. A marked inhibition in DNA fragmentation was observed with a 5–10 nM concentration range of IGF-1. DNA degradation was almost completely blocked in the presence of 50 nM IGF-1. Insulin also exhibited a similar anti-apoptotic effect
(Fig. 1B) . There was a gradual decrease in apoptosis induced by staurosporine when cells were treated with increasing concentrations of insulin (100–400 nM). The percentage of apoptosis-positive cells detected by Hoechst staining was significantly reduced in the presence of IGF-1 and insulin
(Figs. 1C and 1D) . Approximately 40% to 50% of the cells in cultures treated with staurosporine showed the characteristic intense blue nuclear stain due to DNA condensation. Whereas the presence of 10 nM IGF-1 or 400 nM insulin decreased the number of apoptotic cells to approximately 16%, only approximately 3% of cells were found to be apoptotic with 50 nM IGF-1. The ability of IGF-1 to suppress apoptosis was also examined over a prolonged time period. While staurosporine induced significant change in cuboidal shape of cells, cell detachment, and death due to apoptosis
(Fig. 2) , IGF-1 was able to rescue the lens epithelial cells from apoptotic cell death even after 30 hours (
Figs. 2A and 2B , right panel). A diffuse appearance of cellular contents with no clear boundaries between the cells and pyknosis of nuclei without distinct shape were the morphologic changes observed when staurosporine was present in the cultures (
Fig. 2B , middle panel). The presence of IGF-1 blocked the staurosporine effect on cell morphology, which remained similar to that of control cells not treated with staurosporine. Very few cells were detached in these samples after 30 hours. In staurosporine-treated cultures there was an increase in DNA breakdown with time (
Fig. 2A , left and middle panels), and after 30 hours, low-molecular-weight DNA fragments disappeared due to increased breakdown (
Fig. 2A , right panel). In the presence of IGF-1 (50 nM), very little DNA fragmentation was detected after a 30-hour period (
Fig. 2A , right panel).